Immunomodulatory effects of vitamin D on TLR7 and TLR8 signalling in monocytes and monocyte-derived macrophages

Abstract

Monocytes and macrophages are crucial innate immune cells known for their role in antiviral signalling. They respond to viruses via intracellular membrane receptors such as toll-like receptor 7 (TLR7) and toll-like receptor 8 (TLR8). When these receptors are activated, inflammatory cytokines and chemokines are produced downstream which play an important role in the inhibition of viral replication. However, the excessive production of these proinflammatory mediators results in chronic inflammation which can cause multi-organ failure and has been linked to various autoimmune disorders. Thus, there is a need to regulate these pathways. 1,25-dihydroxyvitamin D3 (1,25D3) is a recognised immune modulator, widely accepted as promoting an anti-inflammatory state. 1,25D3 is thought to reduce the production of proinflammatory mediators, thereby playing a role in the maintenance of immune homeostasis. As such, the aim of this research project was to investigate the effect of 1,25D3 on TLR7 and TLR8 expression and function in monocytes and monocyte-derived macrophages (MDMs). This was done by assessing the expression of TLR7 and TLR8 in Tohoku Hospital Pediatrics-1 cells (THP-1) and THP-1 derived macrophages, and the effect of 1,25D3 thereon using RNA-sequencing. Furthermore, interleukin-1 beta (IL1β), chemokine (C-C motif) ligand 2 (CCL2), C-X-C motif chemokine ligand 10 (CXCL10), and Interleukin-10 (IL10) expression was measured using RT-qPCR in monocytes and MDMs cultured in the absence or presence of 1,25D3. Thereafter, the downstream functional effects were investigated by activating the TLR7 and TLR8 signalling pathways using vesatolimod and motolimod, respectively, in monocytes and MDMs cultured in the absence or presence of 1,25D3 and measuring the expression of IL1β, CCL2, CXCL10, and IL10. Lastly, to assess the effect of additional 1,25D3 supplementation, TLR7 and TLR8-stimulated monocytes and MDMs cultured in a 1,25D3 ‘sufficient’ vs 1,25D3 ‘insufficient’ environment were treated with additional 1,25D3, and the expression of IL1β, CCL2, CXCL10, and IL10 were measured using RT-qPCR. Results showed that the expression of TLR7 was decreased, while the expression of TLR8 was increased in both monocytes and MDMs cultured in the presence of 1,25D3. Additionally, the expression of IL1β, CCL2, and CXCL10 were decreased in the MDMs cultured in the presence of 1,25D3. Interestingly, the monocytes responded to TLR8 stimulation, but not TLR7 stimulation, while the MDMs responded to both TLR7 and TLR8 stimulation, regardless of the absence or presence of 1,25D3. The expression IL1β, CCL2, and CXCL10 appeared to be enhanced in monocytes cultured in the presence of 1,25D3, while only CCL2 and CXCL10 expression were enhanced in MDMs cultured in the presence of 1,25D3. However, this trend was not observed with IL1β expression, which was decreased in MDMs cultured in the presence of 1,25D3. Lastly, monocytes and MDMs cultured in a 1,25D3 ‘insufficient’ environment continued to respond to TLR7 and TLR8 stimulation upon additional 1,25D3 supplementation, but while monocytes and MDMs cultured in a 1,25D3 ‘sufficient’ environment continued to respond to TLR8 stimulation, they were no longer responsive to TLR7 stimulation upon additional 1,25D3 supplementation. Overall, these results highlight the role of 1,25D3 as an immunomodulator, with its ability to both enhance the production of proinflammatory mediators, while also modulating their production to prevent the risk of creating a hyperinflammatory environment. This research directly demonstrated the contrasting effects of 1,25D3 on TLR7 and TLR8 signalling which not many studies have highlighted. Additionally, this study aimed to highlight the benefits and importance of maintaining sufficient 1,25D3 levels.